1 1906 140 ENHANCER OF ZESTE HOMOLOG 2-CATALYSED H3K27 TRIMETHYLATION PLAYS A KEY ROLE IN ACUTE-ON-CHRONIC LIVER FAILURE VIA TNF-MEDIATED PATHWAY. ACUTE-ON-CHRONIC LIVER FAILURE IS MAINLY DUE TO HOST IMMUNITY SELF-DESTRUCTION. THE HISTONE H3 LYSINE 27 (H3K27) TRIMETHYLATING ENZYME, ENHANCER OF ZESTE HOMOLOG 2 (EZH2) MEDIATES EPIGENETIC SILENCING OF GENE EXPRESSION AND REGULATES IMMUNITY, ALSO INVOLVES PATHOGENESIS OF SEVERAL LIVER DISEASES. THE CURRENT STUDY WAS TO DETERMINE THE ROLE OF METHYLTRANSFERASE EZH2 AND ITS CATALYSED H3K27 TRIMETHYLATION (H3K27ME3) IN LIVER FAILURE, AND TO FURTHER INVESTIGATE THE POTENTIAL TARGET FOR LIVER FAILURE TREATMENT. EZH2 AND ITS CATALYSED H3K27ME3 WERE DETERMINED IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM LIVER FAILURE PATIENTS AND KUPFFER CELLS FROM EXPERIMENTAL MICE. FURTHERMORE, GSK126 (AN INHIBITOR FOR EZH2 TRIMETHYLATION FUNCTION) WAS APPLIED IN LIVER FAILURE MICE IN VIVO, AND LIPOPOLYSACCHARIDE-STIMULATED MONONUCLEAR CELLS IN VITRO. EZH2 AND H3K27ME3 WERE SIGNIFICANTLY UPREGULATED IN HUMAN PBMC FROM LIVER FAILURE PATIENTS OR MURINE KUPFFER CELLS FROM THE LIVER FAILURE ANIMALS, RESPECTIVELY. GSK126 AMELIORATED DISEASE SEVERITY IN LIVER FAILURE MICE, WHICH MAYBE ATTRIBUTE TO DOWN-REGULATE CIRCULATING AND HEPATIC PROINFLAMMATORY CYTOKINES, ESPECIALLY TNF VIA REDUCING H3K27ME3. IN-DEPTH CHROMATIN IMMUNOPRECIPITATION ANALYSIS UNRAVELLED THAT DECREASED ENRICHMENT OF H3K27ME3 ON TNF PROMOTOR, RESULTING IN TNF ELEVATION IN KUPFFER CELLS FROM LIVER FAILURE MICE. NUCLEAR FACTOR KAPPA B (NF-KAPPAB) AND PROTEIN KINASE B (AKT) SIGNALLING PATHWAYS WERE ACTIVATED UPON LIPOPOLYSACCHARIDE STIMULATION, BUT ATTENUATED BY USING GSK126, ACCOMPANIED WITH DECREASED TNF IN VITRO. IN CONCLUSION, EZH2 AND H3K27ME3 CONTRIBUTED TO THE PATHOGENESIS OF LIVER FAILURE VIA TRIGGERING TNF AND OTHER INDISPENSABLE PROINFLAMMATORY CYTOKINES. EZH2 WAS TO MODIFY H3K27ME3 ENRICHMENT, AS WELL AS, ACTIVATION OF THE DOWNSTREAM NF-KAPPAB AND AKT SIGNALLING PATHWAYS. 2018 2 5319 40 PTEN IS FUNDAMENTAL FOR ELIMINATION OF LEUKEMIA STEM CELLS MEDIATED BY GSK126 TARGETING EZH2 IN CHRONIC MYELOGENOUS LEUKEMIA. PURPOSE: LEUKEMIA STEM CELLS (LSCS) ARE AN IMPORTANT SOURCE OF TYROSINE KINASE INHIBITOR RESISTANCE AND DISEASE RELAPSE IN PATIENTS WITH CHRONIC MYELOGENOUS LEUKEMIA (CML). TARGETING LSCS MAY BE AN ATTRACTIVE STRATEGY TO OVERRIDE THIS THORNY PROBLEM. GIVEN THAT EZH2 WAS OVEREXPRESSED IN PRIMARY CML CD34(+) CELLS, OUR PURPOSE IN THIS STUDY WAS TO EVALUATE THE EFFECTS OF TARGETING EZH2 ON CML LSCS AND CLARIFY ITS UNDERLYING MECHANISM.EXPERIMENTAL DESIGN: HUMAN PRIMARY CML CD34(+) CELLS AND RETROVIRALLY BCR-ABL-DRIVEN CML MOUSE MODELS WERE EMPLOYED TO EVALUATE THE EFFECTS OF SUPPRESSION OF EZH2 BY GSK126- OR EZH2-SPECIFIC SHRNA IN VITRO AND IN VIVO RECRUITMENT OF EZH2 AND H3K27ME3 ON THE PROMOTER OF TUMOR-SUPPRESSOR GENE PTEN IN CML CELLS WAS MEASURED BY CHROMATIN IMMUNOPRECIPITATION ASSAY.RESULTS: OUR RESULTS SHOWED THAT PHARMACOLOGIC INHIBITION OF EZH2 BY GSK126 NOT ONLY ELICITED APOPTOSIS AND RESTRICTED CELL GROWTH IN CML BULK LEUKEMIA CELLS, BUT ALSO DECREASED LSCS IN CML CD34(+) CELLS WHILE SPARING THOSE FROM NORMAL BONE MARROW CD34(+) CELLS. SUPPRESSION OF EZH2 BY GSK126 OR SPECIFIC SHRNA PROLONGED SURVIVAL OF CML MICE AND REDUCED THE NUMBER OF LSCS IN MICE. EZH2 KNOCKDOWN RESULTED IN ELEVATION OF PTEN AND LED TO IMPAIRED RECRUITMENT OF EZH2 AND H3K27ME3 ON THE PROMOTER OF PTEN GENE. THE EFFECT OF EZH2 KNOCKDOWN IN THE CML MICE WAS AT LEAST PARTIALLY REVERSED BY PTEN KNOCKDOWN.CONCLUSIONS: THESE FINDINGS IMPROVE THE UNDERSTANDING OF THE EPIGENETIC REGULATION OF STEMNESS IN CML LSCS AND WARRANT CLINICAL TRIAL OF GSK126 IN REFRACTORY PATIENTS WITH CML. CLIN CANCER RES; 24(1); 145-57. (C)2017 AACR. 2018 3 2326 41 EPIGENETIC REGULATION OF HOTAIR IN ADVANCED CHRONIC MYELOID LEUKEMIA. PURPOSE: CHRONIC MYELOID LEUKEMIA (CML) ACCOUNTS FOR ~10% OF LEUKEMIA CASES, AND ITS PROGRESSION INVOLVES EPIGENETIC GENE REGULATION. THIS STUDY INVESTIGATED EPIGENETIC REGULATION OF HOTAIR AND ITS TARGET MICRORNA, MIR-143, IN ADVANCED CML. PATIENTS AND METHODS: WE FIRST ISOLATED BONE MARROW MONONUCLEAR CELLS FROM 70 PATIENTS WITH DIFFERENT PHASES OF CML AND FROM HEALTHY DONORS AS NORMAL CONTROL; WE ALSO CULTURED K562 AND KCL22 CELLS, TREATED WITH DEMETHYLATION DRUG; MTT ASSAY, FLOW CYTOMETRY, QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), WESTERN BLOT, LUCIFERASE ASSAY, RNA PULL-DOWN ASSAY AND RNA-BINDING PROTEIN IMMUNOPRECIPITATION (RIP) ASSAY WERE PERFORMED. RESULT: AS MEASURED BY QPCR, HOTAIR EXPRESSION IN K562 CELLS, KCL22 CELLS, AND SAMPLES FROM CASES OF ADVANCED-STAGE CML INCREASED WITH LEVELS OF SEVERAL DNA METHYLTRANSFERASES AND HISTONE DEACETYLATES, INCLUDING DNMT1, DNMT3A, HDAC1, EZH2, AND LSD1, AND MIR-143 LEVELS WERE DECREASED AND HOTAIR LEVELS WERE INCREASED. TREATMENT WITH 5-AZACYTIDINE, A DNA METHYLATION INHIBITOR, DECREASED DNMT1, DNMT3A, HDAC1, EZH2, LSD1 MRNA, PROTEIN LEVELS, AND HOTAIR MRNA LEVELS BUT INCREASED MIR-143 LEVELS. HOTAIR KNOCKDOWN AND MIR-143 OVEREXPRESSION BOTH INHIBITED PROLIFERATION AND PROMOTED APOPTOSIS IN KCL22 AND K562 CELLS THROUGH THE PI3K/AKT PATHWAY. RNA PULL-DOWN, MASS SPECTROMETRY, AND RIP ASSAYS SHOWED THAT HOTAIR INTERACTED WITH EZH2 AND LSD1. A DUAL-LUCIFERASE ASSAY DEMONSTRATED THAT HOTAIR INTERACTED WITH MIR-143. CONCLUSION: OUR FINDINGS DEMONSTRATE THE KEY EPIGENETIC INTERACTIONS OF HOTAIR RELATED TO CML PROGRESSION AND SUGGEST HOTAIR AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AS A CML TREATMENT STRATEGY. 2018 4 2784 39 EZH2 PROMOTES EXTRACELLULAR MATRIX DEGRADATION VIA NUCLEAR FACTOR-KAPPAB (NF-KAPPAB) AND P38 SIGNALING PATHWAYS IN PULPITIS. PULPITIS IS A COMPLICATED CHRONIC INFLAMMATORY PROCESS WHICH CAN BE IN A DYNAMIC BALANCE BETWEEN DAMAGE AND REPAIR. THE EXTRACELLULAR MATRIX PLAYS AN IMPORTANT REGULATORY ROLE IN WOUND HEALING AND TISSUE REPAIR. THE AIM OF THIS STUDY WAS TO EXPLORE THE ROLE OF THE EPIGENETIC MARK, ENHANCER OF ZESTE HOMOLOG 2 (EZH2) ON THE DEGRADATION OF EXTRACELLULAR MATRIX DURING PULPITIS. QUANTITATIVE POLYMERASE CHAIN REACTION WAS USED TO ASSESS THE EXPRESSION OF MATRIX METALLOPROTEINASES (MMPS) AND TYPE I COLLAGEN IN HUMAN DENTAL PULP CELLS (HDPCS) UPON EZH2 AND EI1 (EZH2 INHIBITOR) STIMULATION. THE MECHANISM OF EZH2 AFFECTING EXTRACELLULAR MATRIX WAS EXPLORED THROUGH QUANTITATIVE POLYMERASE CHAIN REACTION AND WESTERN BLOT. A RAT MODEL OF DENTAL PULP INFLAMMATION WAS ESTABLISHED, AND THE EXPRESSION OF TYPE I COLLAGEN IN DENTAL PULP UNDER EZH2 STIMULATION WAS DETECTED BY IMMUNOHISTOCHEMICAL STAINING. EZH2 UPREGULATED THE EXPRESSION OF MMP-1, MMP-3, MMP-8, AND MMP-10 AND DECREASED THE PRODUCTION OF TYPE I COLLAGEN IN HDPCS, WHILE EI1 HAD THE OPPOSITE EFFECT. EZH2 ACTIVATED THE NUCLEAR FACTOR-KAPPA B (NF-KAPPAB) AND P38 SIGNALING PATHWAYS IN HDPCS, THE INHIBITION OF WHICH REVERSED THE INDUCTION OF MMPS AND THE SUPPRESSION OF TYPE I COLLAGEN. EZH2 CAN DOWNREGULATE THE TYPE I COLLAGEN LEVELS IN AN EXPERIMENTAL MODEL OF DENTAL PULPITIS IN RATS. EZH2 PROMOTES EXTRACELLULAR MATRIX DEGRADATION VIA NUCLEAR FACTOR-KAPPAB (NF-KAPPAB) AND P38 SIGNALING PATHWAYS IN PULPITIS. EZH2 CAN DECREASE THE TYPE I COLLAGEN LEVELS IN VIVO AND IN VITRO. 2021 5 1905 48 ENHANCER OF ZESTE HOMOLOG 2 CONTRIBUTES TO APOPTOSIS BY INACTIVATING JANUS KINASE 2/ SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION SIGNALING IN INFLAMMATORY BOWEL DISEASE. BACKGROUND: INFLAMMATORY BOWEL DISEASE (IBD) IS A PREVALENT WORLDWIDE HEALTH PROBLEM FEATURED BY RELAPSING, CHRONIC GASTROINTESTINAL INFLAMMATION. ENHANCER OF ZESTE HOMOLOG 2 (EZH2) IS A CRITICAL EPIGENETIC REGULATOR IN DIFFERENT PATHOLOGICAL MODELS, SUCH AS CANCER AND INFLAMMATION. HOWEVER, THE ROLE OF EZH2 IN THE IBD DEVELOPMENT IS STILL OBSCURE. AIM: TO EXPLORE THE EFFECT OF EZH2 ON IBD PROGRESSION AND THE UNDERLYING MECHANISM. METHODS: THE IBD MOUSE MODEL WAS CONDUCTED BY ADDING DEXTRAN SODIUM SULFATE (DSS), AND THE EFFECT OF EZH2 ON DSS-INDUCED COLITIS WAS ASSESSED IN THE MODEL. THE FUNCTION OF EZH2 IN REGULATING APOPTOSIS AND PERMEABILITY WAS EVALUATED BY ANNEXIN V-FITC APOPTOSIS DETECTION KIT, TRANSEPITHELIAL ELECTRICAL RESISTANCE ANALYSIS, AND WESTERN BLOT ANALYSIS OF RELATED MARKERS, INCLUDING ZONA OCCLUDENS 1, CLAUDIN-5, AND OCCLUDIN, IN NCM460 AND FETAL HUMAN COLON (FHC) CELLS. THE MECHANICAL INVESTIGATION WAS PERFORMED BY QUANTITATIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION, WESTERN BLOT ANALYSIS, AND CHROMATIN IMMUNOPRECIPITATION ASSAYS. RESULTS: THE COLON LENGTH WAS INHIBITED IN THE DSS-TREATED MICE AND WAS ENHANCED BY THE EZH2 DEPLETION IN THE SYSTEM. DSS TREATMENT CAUSED A DECREASED HISTOLOGICAL SCORE IN THE MICE, WHICH WAS REVERSED BY EZH2 DEPLETION. THE INFLAMMATORY CYTOKINES, SUCH AS TUMOR NECROSIS FACTOR-ALPHA, INTERLEUKIN-6, AND INTERLEUKIN-1BETA, WERE INDUCED IN THE DSS-TREATED MICE, IN WHICH THE DEPLETION OF EZH2 COULD REVERSE THIS EFFECT. MOREOVER, THE TUMOR NECROSIS FACTOR-ALPHA TREATMENT INDUCED THE APOPTOSIS OF NCM460 AND FHC CELLS, IN WHICH EZH2 DEPLETION COULD REVERSE THIS EFFECT IN THE CELLS. MOREOVER, THE DEPLETION OF EZH2 ATTENUATED PERMEABILITY OF COLONIC EPITHELIAL CELLS. MECHANICALLY, THE DEPLETION OF EZH2 OR EZH2 INHIBITOR GSK343 WAS ABLE TO ENHANCE THE EXPRESSION AND THE PHOSPHORYLATION OF JANUS KINASE 2 (JK2) AND SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION IN THE NCM460 AND FHC CELLS. SPECIFICALLY, EZH2 INACTIVATED JAK2 EXPRESSION BY REGULATING HISTONE H3K27ME3. JAK2 INHIBITOR TG101348 WAS ABLE TO REVERSE EZH2 KNOCKDOWN-MEDIATED COLONIC EPITHELIAL CELL PERMEABILITY AND APOPTOSIS. CONCLUSION: THUS, WE CONCLUDED THAT EZH2 CONTRIBUTED TO APOPTOSIS AND INFLAMMATORY RESPONSE BY INACTIVATING JAK2/ SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION SIGNALING IN IBD. EZH2 MAY BE APPLIED AS A POTENTIAL TARGET FOR IBD THERAPY. 2021 6 2825 53 FLOW-DEPENDENT EPIGENETIC REGULATION OF IGFBP5 EXPRESSION BY H3K27ME3 CONTRIBUTES TO ENDOTHELIAL ANTI-INFLAMMATORY EFFECTS. RATIONALE: ATHEROSCLEROSIS IS A CHRONIC INFLAMMATORY AND EPIGENETIC DISEASE THAT IS INFLUENCED BY DIFFERENT PATTERNS OF BLOOD FLOW. HOWEVER, THE EPIGENETIC MECHANISM WHEREBY ATHEROPROTECTIVE FLOW CONTROLS ENDOTHELIAL GENE PROGRAMMING REMAINS ELUSIVE. HERE, WE INVESTIGATED THE POSSIBILITY THAT FLOW ALTERS ENDOTHELIAL GENE EXPRESSION THROUGH EPIGENETIC MECHANISMS. METHODS: EN FACE STAINING AND WESTERN BLOT WERE USED TO DETECT PROTEIN EXPRESSION. REAL-TIME PCR WAS USED TO DETERMINE RELATIVE GENE EXPRESSION. RNA-SEQUENCING OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS TREATED WITH SIRNA OF ENHANCER OF ZESTE HOMOLOG 2 (EZH2) OR LAMINAR FLOW WAS USED FOR TRANSCRIPTIONAL PROFILING. RESULTS: WE FOUND THAT TRIMETHYLATION OF HISTONE 3 LYSINE 27 (H3K27ME3), A REPRESSIVE EPIGENETIC MARK THAT ORCHESTRATES GENE REPRESSION, WAS REDUCED IN LAMINAR FLOW AREAS OF MOUSE AORTA AND FLOW-TREATED HUMAN ENDOTHELIAL CELLS. THE DECREASE OF H3K27ME3 PARALLELED A REDUCTION IN THE EPIGENETIC "WRITER"-EZH2, THE CATALYTIC SUBUNIT OF THE POLYCOMB REPRESSIVE COMPLEX 2 (PRC2). MOREOVER, LAMINAR FLOW DECREASED EXPRESSION OF EZH2 VIA MECHANOSENSITIVE MIR101. GENOME-WIDE TRANSCRIPTOME PROFILING STUDIES IN ENDOTHELIAL CELLS TREATED WITH EZH2 SIRNA AND FLOW REVEALED THE UPREGULATION OF NOVEL MECHANOSENSITIVE GENE IGFBP5 (INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 5), WHICH IS EPIGENETICALLY SILENCED BY H3K27ME3. FUNCTIONALLY, INHIBITION OF H3K27ME3 BY EZH2 SIRNA OR GSK126 (A SPECIFIC EZH2 INHIBITOR) REDUCED H3K27ME3 LEVELS AND MONOCYTE ADHESION TO ENDOTHELIAL CELLS. ADENOVIRAL OVEREXPRESSION OF IGFBP5 ALSO RECAPITULATED THE ANTI-INFLAMMATORY EFFECTS OF H3K27ME3 INHIBITION. MORE IMPORTANTLY, WE OBSERVED EZH2 UPREGULATION, AND IGFBP5 DOWNREGULATION, IN ADVANCED ATHEROSCLEROTIC PLAQUES FROM HUMAN PATIENTS. CONCLUSION: TAKEN TOGETHER, OUR FINDINGS REVEAL THAT ATHEROPROTECTIVE FLOW REDUCES H3K27ME3 AS A CHROMATIN-BASED MECHANISM TO AUGMENT THE EXPRESSION OF GENES THAT CONFER AN ANTI-INFLAMMATORY RESPONSE IN THE ENDOTHELIUM. OUR STUDY EXEMPLIFIES FLOW-DEPENDENT EPIGENETIC REGULATION OF ENDOTHELIAL GENE EXPRESSION, AND ALSO SUGGESTS THAT TARGETING THE EZH2/H3K27ME3/IGFBP5 PATHWAY MAY OFFER NOVEL THERAPEUTICS FOR INFLAMMATORY DISORDERS SUCH AS ATHEROSCLEROSIS. 2018 7 3531 33 IMATINIB CAUSES EPIGENETIC ALTERATIONS OF PTEN GENE VIA UPREGULATION OF DNA METHYLTRANSFERASES AND POLYCOMB GROUP PROTEINS. WE HAVE RECENTLY REPORTED THE POSSIBLE IMATINIB-RESISTANT MECHANISM; LONG-TERM EXPOSURE OF LEUKEMIA CELLS TO IMATINIB DOWNREGULATED LEVELS OF PHOSPHATASE AND TENSIN HOMOLOG DELETED ON CHROMOSOME 10 (PTEN) VIA HYPERMETHYLATION OF ITS PROMOTER REGION (LEUKEMIA 2010; 24: 1631). THE PRESENT STUDY EXPLORED THE MOLECULAR MECHANISMS BY WHICH IMATINIB CAUSED METHYLATION ON THE PROMOTER REGION OF THIS TUMOR SUPPRESSOR GENE IN LEUKEMIA CELLS. REAL-TIME REVERSE TRANSCRIPTION PCR FOUND THAT LONG-TERM EXPOSURE OF CHRONIC EOSINOPHILIC LEUKEMIA EOL-1 CELLS EXPRESSING FIP1L1/PLATELET-DERIVED GROWTH FACTOR RECEPTOR-ALPHA TO IMATINIB INDUCED EXPRESSION OF DNA METHYLTRANSFERASE 3A (DNMT3A) AND HISTONE-METHYLTRANSFERASE ENHANCER OF ZESTE HOMOLOG 2 (EZH2), A FAMILY OF POLYCOMB GROUP, THEREBY INCREASING METHYLATION OF THE GENE. IMMUNOPRECIPITATION ASSAY FOUND THE INCREASED COMPLEX FORMATION OF DNMT3A AND EZH2 PROTEINS IN THESE CELLS. MOREOVER, CHROMATIN IMMUNOPRECIPITATION ASSAY SHOWED THAT AMOUNTS OF BOTH DNMT3A AND EZH2 PROTEINS BOUND AROUND THE PROMOTER REGION OF PTEN GENE WERE INCREASED IN EOL-1 CELLS AFTER EXPOSURE TO IMATINIB. FURTHERMORE, WE FOUND THAT LEVELS OF DNMT3A AND EZH2 WERE STRIKINGLY INCREASED IN LEUKEMIA CELLS ISOLATED FROM INDIVIDUALS WITH CHRONIC MYELOGENOUS LEUKEMIA (N=1) AND PHILADELPHIA CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (N=2), WHO RELAPSED AFTER TREATMENT WITH IMATINIB COMPARED WITH THOSE ISOLATED AT THEIR INITIAL PRESENTATION. TAKEN TOGETHER, IMATINIB COULD CAUSE DRUG-RESISTANCE VIA RECRUITMENT OF POLYCOMB GENE COMPLEX TO THE PROMOTER REGION OF THE PTEN AND DOWNREGULATION OF THIS GENE'S TRANSCRIPTS IN LEUKEMIA PATIENTS. 2011 8 2425 27 EPIGENETIC SILENCING OF IRF1 DYSREGULATES TYPE III INTERFERON RESPONSES TO RESPIRATORY VIRUS INFECTION IN EPITHELIAL TO MESENCHYMAL TRANSITION. CHRONIC OXIDATIVE INJURY PRODUCED BY AIRWAY DISEASE TRIGGERS A TRANSFORMING GROWTH FACTOR-BETA (TGF-BETA)-MEDIATED EPIGENETIC REPROGRAMMING KNOWN AS THE EPITHELIAL-MESENCHYMAL TRANSITION (EMT). WE OBSERVE THAT EMT SILENCES PROTECTIVE MUCOSAL INTERFERON (IFN)-I AND III PRODUCTION ASSOCIATED WITH ENHANCED RHINOVIRUS (RV) AND RESPIRATORY SYNCYTIAL VIRUS (RSV) REPLICATION. MESENCHYMAL TRANSITIONED CELLS ARE DEFECTIVE IN INDUCIBLE INTERFERON REGULATORY FACTOR 1 (IRF1) EXPRESSION BY OCCLUDING RELA AND IRF3 ACCESS TO THE PROMOTER. IRF1 IS NECESSARY FOR THE EXPRESSION OF TYPE III IFNS (IFNLS 1 AND 2/3). INDUCED BY THE EMT, ZINC FINGER E-BOX BINDING HOMEOBOX 1 (ZEB1) BINDS AND SILENCES IRF1. ECTOPIC ZEB1 IS SUFFICIENT FOR IRF1 SILENCING, WHEREAS ZEB1 KNOCKDOWN PARTIALLY RESTORES IRF1-IFNL UPREGULATION. ZEB1 SILENCES IRF1 THROUGH THE CATALYTIC ACTIVITY OF THE ENHANCER OF ZESTE 2 POLYCOMB REPRESSIVE COMPLEX 2 SUBUNIT (EZH2), FORMING REPRESSIVE H3K27(ME3) MARKS. WE OBSERVE THAT IRF1 EXPRESSION IS MEDIATED BY ZEB1 DE-REPRESSION, AND OUR STUDY DEMONSTRATES HOW AIRWAY REMODELLING/FIBROSIS IS ASSOCIATED WITH A DEFECTIVE MUCOSAL ANTIVIRAL RESPONSE THROUGH ZEB1-INITIATED EPIGENETIC SILENCING. 2017 9 3388 34 HOMOCYSTEINE INDUCES PODOCYTE APOPTOSIS BY REGULATING MIR-1929-5P EXPRESSION THROUGH C-MYC, DNMT1 AND EZH2. CHRONIC KIDNEY DISEASE (CKD) IS A COMMON AND COMPLEX DISEASE IN KIDNEYS WHICH HAS BEEN ASSOCIATED WITH AN INCREASED RISK OF RENAL CELL CARCINOMA. ELEVATED HOMOCYSTEINE (HCY) LEVELS ARE KNOWN TO INFLUENCE THE DEVELOPMENT AND PROGRESSION OF CKD BY REGULATING PODOCYTE INJURY AND APOPTOSIS. TO INVESTIGATE THE MOLECULAR MECHANISMS TRIGGERED IN PODOCYTES BY HCY, WE USED CBS(+/-) MICE AND OBSERVED THAT HIGHER HCY LEVELS INCREASED THE APOPTOSIS RATE OF PODOCYTES WITH ACCOMPANYING GLOMERULAR DAMAGE. HCY-INDUCED PODOCYTE INJURY AND APOPTOSIS IN CBS(+/-) MICE WAS REGULATED BY INHIBITION OF MICRORNA (MIR)-1929-5P EXPRESSION. OVEREXPRESSION OF MIR-1929-5P IN PODOCYTES INHIBITED APOPTOSIS BY UPREGULATING BCL-2. FURTHERMORE, THE EXPRESSION OF MIR-1929-5P WAS REGULATED BY EPIGENETIC MODIFICATIONS OF ITS PROMOTER. HCY UPREGULATED DNA METHYLTRANSFERASE 1 (DNMT1) AND ENHANCER OF ZESTE HOMOLOG 2 (EZH2) LEVELS, RESULTING IN INCREASED DNA METHYLATION AND H3K27ME3 LEVELS ON THE MIR-1929-5P PROMOTER. ADDITIONALLY, WE OBSERVED THAT C-MYC RECRUITED DNMT1 AND EZH2 TO THE MIR-1929-5P PROMOTER AND SUPPRESSED THE EXPRESSION OF MIR-1929-5P. IN SUMMARY, WE DEMONSTRATED THAT HCY PROMOTES PODOCYTE APOPTOSIS THROUGH THE REGULATION OF THE EPIGENETIC MODIFIERS DNMT1 AND EZH2, WHICH ARE RECRUITED BY C-MYC TO THE PROMOTER OF MIR-1929-5P TO SILENCE MIR-1929-5P EXPRESSION. 2021 10 717 47 CALCITONIN GENE-RELATED PEPTIDE REGULATES SPINAL MICROGLIAL ACTIVATION THROUGH THE HISTONE H3 LYSINE 27 TRIMETHYLATION VIA ENHANCER OF ZESTE HOMOLOG-2 IN RATS WITH NEUROPATHIC PAIN. BACKGROUND: CALCITONIN GENE-RELATED PEPTIDE (CGRP) AS A MEDIATOR OF MICROGLIAL ACTIVATION AT THE TRANSCRIPTIONAL LEVEL MAY FACILITATE NOCICEPTIVE SIGNALING. TRIMETHYLATION OF H3 LYSINE 27 (H3K27ME3) BY ENHANCER OF ZESTE HOMOLOG 2 (EZH2) IS AN EPIGENETIC MARK THAT REGULATES INFLAMMATORY-RELATED GENE EXPRESSION AFTER PERIPHERAL NERVE INJURY. IN THIS STUDY, WE EXPLORED THE RELATIONSHIP BETWEEN CGRP AND H3K27ME3 IN MICROGLIAL ACTIVATION AFTER NERVE INJURY, AND ELUCIDATED THE UNDERLYING MECHANISMS IN THE PATHOGENESIS OF CHRONIC NEUROPATHIC PAIN. METHODS: MICROGLIAL CELLS (BV2) WERE TREATED WITH CGRP AND DIFFERENTIALLY ENRICHMENTS OF H3K27ME3 ON GENE PROMOTERS WERE EXAMINED USING CHIP-SEQ. A CHRONIC CONSTRICTION INJURY (CCI) RAT MODEL WAS USED TO EVALUATE THE ROLE OF CGRP ON MICROGLIAL ACTIVATION AND EZH2/H3K27ME3 SIGNALING IN CCI-INDUCED NEUROPATHIC PAIN. RESULTS: OVEREXPRESSIONS OF EZH2 AND H3K27ME3 WERE CONFIRMED IN SPINAL MICROGLIA OF CCI RATS BY IMMUNOFLUORESCENCE. CGRP TREATMENT INDUCED THE INCREASED OF H3K27ME3 EXPRESSION IN THE SPINAL DORSAL HORN AND CULTURED MICROGLIAL CELLS (BV2) THROUGH EZH2. CHIP-SEQ DATA INDICATED THAT CGRP SIGNIFICANTLY ALTERED H3K27ME3 ENRICHMENTS ON GENE PROMOTERS IN MICROGLIA FOLLOWING CGRP TREATMENT, INCLUDING 173 GAINING H3K27ME3 AND 75 LOSING THIS MARK, WHICH MOSTLY ENRICHED IN REGULATION OF CELL GROWTH, PHAGOSOME, AND INFLAMMATION. QRT-PCR VERIFIED EXPRESSIONS OF REPRESENTATIVE CANDIDATE GENES (TRAF3IP2, BCL2L11, ITGAM, DAB2, NLRP12, WNT3, ADAM10) AND REAL-TIME CELL ANALYSIS (RTCA) VERIFIED MICROGLIAL PROLIFERATION. ADDITIONALLY, CGRP TREATMENT AND CCI INCREASED EXPRESSIONS OF ITGAM, ADAM10, MCP-1, AND CX3CR1, KEY MEDIATORS OF MICROGLIAL ACTIVATION IN SPINAL DORSAL HORN AND CULTURED MICROGLIAL CELLS. SUCH INCREASED EFFECTS INDUCED BY CCI WERE SUPPRESSED BY CGRP ANTAGONIST AND EZH2 INHIBITOR, WHICH WERE CONCURRENTLY ASSOCIATED WITH THE ATTENUATED MECHANICAL AND THERMAL HYPERALGESIA IN CCI RATS. CONCLUSION: OUR FINDINGS HIGHLY INDICATE THAT CGRP IS IMPLICATED IN THE GENESIS OF NEUROPATHIC PAIN THROUGH REGULATING MICROGLIAL ACTIVATION VIA EZH2-MEDIATED H3K27ME3 IN THE SPINAL DORSAL HORN. 2021 11 5940 38 TARGETING METHYLTRANSFERASE PRMT5 ELIMINATES LEUKEMIA STEM CELLS IN CHRONIC MYELOGENOUS LEUKEMIA. IMATINIB-INSENSITIVE LEUKEMIA STEM CELLS (LSCS) ARE BELIEVED TO BE RESPONSIBLE FOR RESISTANCE TO BCR-ABL TYROSINE KINASE INHIBITORS AND RELAPSE OF CHRONIC MYELOGENOUS LEUKEMIA (CML). IDENTIFYING THERAPEUTIC TARGETS TO ERADICATE CML LSCS MAY BE A STRATEGY TO CURE CML. IN THE PRESENT STUDY, WE DISCOVERED A POSITIVE FEEDBACK LOOP BETWEEN BCR-ABL AND PROTEIN ARGININE METHYLTRANSFERASE 5 (PRMT5) IN CML CELLS. OVEREXPRESSION OF PRMT5 WAS OBSERVED IN HUMAN CML LSCS. SILENCING PRMT5 WITH SHRNA OR BLOCKING PRMT5 METHYLTRANSFERASE ACTIVITY WITH THE SMALL-MOLECULE INHIBITOR PJ-68 REDUCED SURVIVAL, SERIAL REPLATING CAPACITY, AND LONG-TERM CULTURE-INITIATING CELLS (LTC-ICS) IN LSCS FROM CML PATIENTS. FURTHER, PRMT5 KNOCKDOWN OR PJ-68 TREATMENT DRAMATICALLY PROLONGED SURVIVAL IN A MURINE MODEL OF RETROVIRAL BCR-ABL-DRIVEN CML AND IMPAIRED THE IN VIVO SELF-RENEWAL CAPACITY OF TRANSPLANTED CML LSCS. PJ-68 ALSO INHIBITED LONG-TERM ENGRAFTMENT OF HUMAN CML CD34+ CELLS IN IMMUNODEFICIENT MICE. MOREOVER, INHIBITION OF PRMT5 ABROGATED THE WNT/BETA-CATENIN PATHWAY IN CML CD34+ CELLS BY DEPLETING DISHEVELLED HOMOLOG 3 (DVL3). THIS STUDY SUGGESTS THAT EPIGENETIC METHYLATION MODIFICATION ON HISTONE PROTEIN ARGININE RESIDUES IS A REGULATORY MECHANISM TO CONTROL SELF-RENEWAL OF LSCS AND INDICATES THAT PRMT5 MAY REPRESENT A POTENTIAL THERAPEUTIC TARGET AGAINST LSCS. 2016 12 574 32 BCR/ABL INCREASES EZH2 LEVELS WHICH REGULATES XIAP EXPRESSION VIA MIRNA-219 IN CHRONIC MYELOID LEUKEMIA CELLS. IN THIS STUDY, WE SHOWED THAT THE LEVELS OF EZH2 IN BONE MARROW MONONUCLEAR CELLS (BMMNCS) ISOLATED FROM INDIVIDUALS WITH CHRONIC MYELOID LEUKEMIA (CML) (N=12) WERE SIGNIFICANTLY GREATER THAN THOSE IN BMMNCS ISOLATED FROM HEALTHY VOLUNTEERS (N=6) AS WELL AS INDIVIDUALS WITH PHILADELPHIA CHROMOSOME-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS. LENTIVIRAL TRANSDUCTION OF THE BCR/ABL GENE IN BA/F3 CELLS INCREASED EZH2 LEVELS IN PARALLEL WITH PHOSPHORYLATION OF STAT5. NOTABLY, CHROMATIN IMMUNOPRECIPITATION ASSAYS SHOWED THAT STAT5A BOUND TO A PROMOTER REGION OF THE EZH2 GENE, RESULTING IN AN INCREASE IN THE TRANSCRIPTIONAL ACTIVITY OF EZH2 IN LEUKEMIA CELLS. IMPORTANTLY, DOWNREGULATION OF EZH2 BY SHORT HAIRPIN RNAS (SHRNAS) INHIBITED THE EXPRESSION OF XIAP AND INCREASED THE MIR-219 LEVELS ASSOCIATED WITH A DECREASE IN HYPERMETHYLATION OF MIR-219-1 CPG ISLANDS. MOREOVER, OVEREXPRESSION OF MIR-219 DECREASED THE LEVELS OF XIAP IN CML CELLS. SINCE THE 3'-UNTRANSLATED REGION (3'-UTR) OF XIAP CONTAINS MIR219-5P-COMPLEMENTARY BINDING SITE, MIR-219 MIGHT MODULATE THE EXPRESSION OF XIAP THROUGH BINDING OF MIR-219 ON THE 3'-UTR OF XIAP. TAKEN TOGETHER, BCR/ABL POSITIVELY REGULATES THE EXPRESSION OF EZH2 VIA STAT5 SIGNALING. EZH2 MODULATES EPIGENETIC CHANGES AT DNA METHYLATED REGIONS ENCODING MIR-219 AND DOWNREGULATES THE LEVEL OF MIR-219, RESULTING IN UPREGULATION OF XIAP. 2016 13 273 49 AGE-INDUCED SUPPRESSION OF EZH2 MEDIATES INJURY OF PODOCYTES BY REDUCING H3K27ME3. BACKGROUND: CHRONIC HYPERGLYCEMIA, A PIVOTAL FEATURE OF DIABETES MELLITUS (DM), INITIATES THE FORMATION OF ADVANCED GLYCATION END PRODUCTS (AGES) AND THE DYSREGULATION OF EPIGENETIC MECHANISMS, WHICH MAY CAUSE INJURY TO RENAL PODOCYTES, A CENTRAL FEATURE OF DIABETIC KIDNEY DISEASE (DKD). PREVIOUS DATA OF OUR GROUP SHOWED THAT AGES SIGNIFICANTLY REDUCE THE EXPRESSION OF NIPP1 (NUCLEAR INHIBITOR OF PROTEIN PHOSPHATASE 1) IN PODOCYTES IN VITRO AS WELL AS IN HUMAN AND MURINE DKD. NIPP1 WAS SHOWN BY OTHERS TO INTERACT WITH ENHANCER OF ZESTE HOMOLOG 2 (EZH2), WHICH CATALYZES THE REPRESSIVE METHYLATION OF H3K27ME3 ON HISTONE 3. THEREFORE, WE HYPOTHESIZED THAT AGES CAN DIRECTLY INDUCE EPIGENETIC CHANGES IN PODOCYTES. METHODS: WE ANALYZED THE RELEVANCE OF AGES ON EZH2 EXPRESSION AND ACTIVITY IN A MURINE PODOCYTE CELL LINE. CELLS WERE TREATED WITH 5 MG/ML GLYCATED BSA FOR 24 H. TO DETERMINE THE MEANING OF EZH2 SUPPRESSION, EZH2 ACTIVITY WAS INHIBITED BY INCUBATING THE CELLS WITH THE PHARMACOLOGICAL METHYLTRANSFERASE INHIBITOR 3-DEAZANEPLANOCIN A; EZH2 EXPRESSION WAS REPRESSED WITH SIRNA. MRNA EXPRESSION WAS ANALYZED WITH REAL-TIME PCR, AND PROTEIN EXPRESSION WITH WESTERN BLOT. EZH2 EXPRESSION AND LEVEL OF H3K27 TRIMETHYLATION IN PODOCYTES OF DIABETIC DB/DB MICE, A MOUSE MODEL FOR TYPE 2 DM, WERE ANALYZED USING IMMUNOFLUORESCENCE. RESULTS: OUR DATA DEMONSTRATED THAT AGES DECREASE EZH2 EXPRESSION IN PODOCYTES AND CONSEQUENTLY REDUCE H3K27ME3. THIS SUPPRESSION OF EZH2 MIMICKED THE AGE EFFECTS AND CAUSED AN UPREGULATED EXPRESSION OF PATHOLOGICAL FACTORS THAT CONTRIBUTE TO PODOCYTE INJURY IN DKD. IN ADDITION, ANALYSES OF DB/DB MICE SHOWED SIGNIFICANTLY REDUCED H3K27ME3 AND EZH2 EXPRESSION IN PODOCYTES. MOREOVER, THE SUPPRESSION OF NIPP1 AND EZH2 SHOWED SIMILAR EFFECTS REGARDING PODOCYTE INJURY. CONCLUSIONS: OUR STUDIES PROVIDE A NOVEL PATHWAY HOW AGES CONTRIBUTE TO PODOCYTE INJURY AND THE FORMATION OF THE SO-CALLED METABOLIC MEMORY IN DKD. 2020 14 5459 35 RESEARCH ON THE EPIGENETIC REGULATION MECHANISM OF THE PTPN6 GENE IN ADVANCED CHRONIC MYELOID LEUKAEMIA. PTPN6, A TYROSINE PHOSPHATASE PROTEIN, PLAYS A NEGATIVE ROLE IN CELL SIGNAL TRANSDUCTION AND IS NEGATIVELY CORRELATED WITH TUMOUR FORMATION AND GROWTH. HOWEVER, EPIGENETIC REGULATION MECHANISM OF THE PTPN6 GENE IN ADVANCED CHRONIC MYELOID LEUKAEMIA (CML) REMAINS UNCLEAR. THIS STUDY INVESTIGATED BONE MARROW OR BLOOD SAMPLES FROM 44 CML PATIENTS AND 10 HEALTHY VOLUNTEERS. KCL22 AND K562 CELLS WERE CULTURED AND TREATED WITH DEMETHYLATION DRUGS AND HISTONE DEACETYLASE INHIBITORS. REAL TIME QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC PCR, BISULFITE SEQUENCING PCR, WESTERN BLOTTING, CO-IMMUNOPRECIPITATION AND CHROMATIN IMMUNOPRECIPITATION (CHIP) WAS PERFORMED. PTPN6 WAS DOWN-REGULATED IN CELL LINES AND PATIENTS WITH ADVANCED PHASE CML, WHEREAS DNMT1, DNMT3A, MECP2, MBD2 AND HDAC1 WERE UP-REGULATED. TREATMENT WITH 5-AZACYTIDINE, DECITABINE, SODIUM VALPROATE AND LBH589 INCREASED PTPN6 EXPRESSION, BUT DECREASED THAT OF DNMT1, DNMT3A, MECP2, MBD2 AND HDAC1. IMMUNOPRECIPITATION AND MASS SPECTROMETRY SHOWED THAT HDAC1 COMBINED DIRECTLY WITH PTPN6. CHIP-SEQ SHOWED THAT HDAC1 DID NOT COMBINE WITH THE PROMOTER REGION OF PTPN6, WHILE MAPK, AKT, STAT5, JAK2 AND MYC PROMOTER REGIONS ALL COMBINED WITH HDAC1. PTPN6 IS ASSOCIATED WITH PROGRESSION OF CML. LOW EXPRESSION LEVEL OF PTPN6 WAS ASSOCIATED WITH DNA METHYLATION AND REGULATED BY HISTONE ACETYLATION. HDAC1 PARTICIPATES IN THE REGULATION OF PTPN6. 2017 15 3877 38 KDM6A PROMOTES IMATINIB RESISTANCE THROUGH YY1-MEDIATED TRANSCRIPTIONAL UPREGULATION OF TRKA INDEPENDENTLY OF ITS DEMETHYLASE ACTIVITY IN CHRONIC MYELOGENOUS LEUKEMIA. RATIONALE: DESPITE LANDMARK THERAPY OF CHRONIC MYELOGENOUS LEUKEMIA (CML) WITH TYROSINE KINASE INHIBITORS (TKIS), DRUG RESISTANCE REMAINS PROBLEMATIC. CANCER PATHOGENESIS INVOLVES EPIGENETIC DYSREGULATION AND IN PARTICULAR, HISTONE LYSINE DEMETHYLASES (KDMS) HAVE BEEN IMPLICATED IN TKI RESISTANCE. WE SOUGHT TO IDENTIFY KDMS WITH ALTERED EXPRESSION IN CML AND DEFINE THEIR CONTRIBUTION TO IMATINIB RESISTANCE. METHODS: BIOINFORMATICS SCREENING COMPARED KDM EXPRESSION IN CML VERSUS NORMAL BONE MARROW WITH SHRNA KNOCKDOWN AND FLOW CYTOMETRY USED TO MEASURE EFFECTS ON IMATINIB-INDUCED APOPTOSIS IN K562 CELLS. TRANSCRIPTOMIC ANALYSES WERE PERFORMED AGAINST KDM6A CRISPR KNOCKOUT/SHRNA KNOCKDOWN K562 CELLS ALONG WITH GENE RESCUE EXPERIMENTS USING WILDTYPE AND MUTANT DEMETHYLASE-DEAD KDM6A CONSTRUCTS. CO-IMMUNOPRECIPITATION, LUCIFERASE REPORTER AND CHIP WERE EMPLOYED TO ELUCIDATE MECHANISMS OF KDM6A-DEPENDENT RESISTANCE. RESULTS: AMONGST FIVE KDMS UPREGULATED IN CML, ONLY KDM6A DEPLETION SENSITIZED CML CELLS TO IMATINIB-INDUCED APOPTOSIS. RE-INTRODUCTION OF DEMETHYLASE-DEAD KDM6A AS WELL AS WILD-TYPE KDM6A RESTORED IMATINIB RESISTANCE. RNA-SEQ IDENTIFIED NTRK1 GENE DOWNREGULATION AFTER DEPLETION OF KDM6A. MOREOVER, NTRK1 EXPRESSION POSITIVELY CORRELATED WITH KDM6A IN A SUBSET OF CLINICAL CML SAMPLES AND KDM6A KNOCKDOWN IN FRESH CML ISOLATES DECREASED NTRK1 ENCODED PROTEIN (TRKA) EXPRESSION. MECHANISTICALLY, KDM6A WAS RECRUITED TO THE NTRK1 PROMOTER BY THE TRANSCRIPTION FACTOR YY1 WITH SUBSEQUENT TRKA UPREGULATION ACTIVATING DOWN-STREAM SURVIVAL PATHWAYS TO INVOKE IMATINIB RESISTANCE. CONCLUSION: CONTRARY TO ITS REPORTED ROLE AS A TUMOR SUPPRESSOR AND INDEPENDENT OF ITS DEMETHYLASE FUNCTION, KDM6A PROMOTES IMATINIB-RESISTANCE IN CML CELLS. THE IDENTIFICATION OF THE KDM6A/YY1/TRKA AXIS AS A NOVEL IMATINIB-RESISTANCE MECHANISM REPRESENTS AN UNEXPLORED AVENUE TO OVERCOME TKI RESISTANCE IN CML. 2021 16 5458 34 RESEARCH ON EPIGENETIC MECHANISM OF SFRP2 IN ADVANCED CHRONIC MYELOID LEUKEMIA. SECRETED FRIZZLED-RELATED PROTEIN 2 (SFRP2) HAS BEEN REPORTED TO ACT AS A TUMOR SUPPRESSORS. THIS STUDY AIMS TO DETECT THE BIOLOGICAL ROLE OF SFRP2 IN ADVANCED CHRONIC MYELOID LEUKEMIA (CML). IN THIS STUDY WE EXAMINED BONE MARROW SAMPLES FROM 45 CML PATIENTS AND 10 HEALTHY DONORS. K562 AND KCL22 CELLS WERE CULTURED AND TREATED WITH DEMETHYLATION DRUG AND HISTONE DEACETYLASE INHIBITOR (HDACI). KCL22 AND K562 CELLS WERE TRANSFECTED WITH LENTIVIRAL VECTOR (LV)-SFRP2, LV-CONTROL. THE CELLS WERE THEN SUBJECTED TO PROLIFERATION AND APOPTOSIS ASSAYS, REAL TIME POLYMERASE CHAIN REACTION (PCR), METHYLATION-SPECIFIC PCR (MSP), WESTERN BLOTTING, CO-IMMUNOPRECIPITATION (COIP) AND CHROMATIN IMMUNOPRECIPITATION (CHIP), WE FOUND THAT SFRP2 WAS DOWN-REGULATED IN THE ACCELERATED AND BLAST PHASE OF CML, WHEREAS, THE LEVELS OF WNT1, WNT3 AND WNT5A WERE UP-REGULATED IN THE ACCELERATED AND BLAST PHASE OF CML. OVEREXPRESSION SFRP2 INHIBITED PROLIFERATION, PROMOTED APOPTOSIS AND ACTIVATED THE WNT PATHWAY. COIP-MS RESULTS SHOWED THAT SFRP2 INTERACTED WITH WNT1 AND WNT5A. CHIP-SEQ RESULT INDICATED THAT THE PROMOTER OF H3K4ME3 AND H3K27ME3 WERE ABLE TO INTERACT WITH SFRP2. IN CONCLUSION, OUR FINDINGS DEMONSTRATED THE SFRP2 ACT AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AND HDACI AS A POTENTIAL CML TREATMENT STRATEGY. 2018 17 2748 47 EXPRESSION AND FUNCTION OF EZH2 IN SYNOVIAL FIBROBLASTS: EPIGENETIC REPRESSION OF THE WNT INHIBITOR SFRP1 IN RHEUMATOID ARTHRITIS. OBJECTIVES: TO STUDY THE EXPRESSION, REGULATION AND FUNCTION OF THE HISTONE METHYLTRANSFERASE ENHANCER OF ZESTE HOMOLOGUE 2 (EZH2) IN SYNOVIAL FIBROBLASTS (SF) FROM PATIENTS WITH RHEUMATOID ARTHRITIS (RA) AND OSTEOARTHRITIS (OA). METHODS: SF WERE OBTAINED FROM RA AND OA PATIENTS UNDERGOING JOINT SURGERY. EXPRESSION LEVELS WERE ASSESSED BY QUANTITATIVE REAL-TIME PCR AND WESTERN BLOT. KINASE INHIBITORS AND REPORTER GENE ASSAYS WERE EMPLOYED TO STUDY SIGNALLING PATHWAYS. FUNCTIONAL ANALYSES INCLUDED EZH2 OVEREXPRESSION BY PLASMID TRANSFECTION AND GENE SILENCING BY SMALL INTERFERING RNA. CHROMATIN IMMUNOPRECIPITATION ASSAY WAS USED TO ANALYSE HISTONE METHYLATION WITHIN DISTINCT PROMOTER REGIONS. RESULTS: BY STUDYING THE EXPRESSION AND FUNCTION OF EZH2 IN SF THE AUTHORS FOUND THAT EZH2 IS OVEREXPRESSED IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS (RASF) AND FURTHER INDUCED BY TUMOUR NECROSIS FACTOR ALPHA THROUGH THE NUCLEAR FACTOR KAPPA B AND JUN KINASE PATHWAYS. AS A TARGET GENE OF EZH2 THE AUTHORS IDENTIFIED SECRETED FRIZZLED-RELATED PROTEIN 1 (SFRP1), AN INHIBITOR OF WNT SIGNALLING, WHICH IS ASSOCIATED WITH THE ACTIVATION OF RASF, AND SHOW THAT SFRP1 EXPRESSION CORRELATES WITH THE OCCUPATION OF ITS PROMOTER WITH ACTIVATING AND SILENCING HISTONE MARKS. CONCLUSIONS: THESE DATA STRONGLY SUGGEST THAT THE CHRONIC INFLAMMATORY ENVIRONMENT OF THE RA JOINT INDUCES EZH2 AND THUS MIGHT CAUSE CHANGES IN THE EPIGENETIC PROGRAMMES OF SF. 2011 18 5479 38 RESVERATROL ATTENUATES CIGARETTE SMOKE EXTRACT INDUCED CELLULAR SENESCENCE IN HUMAN AIRWAY EPITHELIAL CELLS BY REGULATING THE MIR-34A/SIRT1/NF-KAPPAB PATHWAY. CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) IS CHARACTERIZED BY ACCELERATED LUNG AGING. SMOKING IS THE CRITICAL RISK FACTOR FOR COPD. CELLULAR SENESCENCE OF AIRWAY EPITHELIAL CELLS IS THE CYTOLOGICAL BASIS OF ACCELERATED LUNG AGING IN COPD, AND THE REGULATION OF MICRORNAS (MIRNAS) IS THE CENTRAL EPIGENETIC MECHANISM OF CELLULAR SENESCENCE. RESVERATROL (RES) IS A POLYPHENOL WITH ANTI-AGING PROPERTIES. THIS STUDY INVESTIGATED WHETHER RES ATTENUATES CIGARETTE SMOKE EXTRACT (CSE)-INDUCED CELLULAR SENESCENCE IN HUMAN AIRWAY EPITHELIAL CELLS (BEAS-2B) THROUGH THE MIR-34A/SIRT1/NUCLEAR FACTOR-KAPPAB (NF-KAPPAB) PATHWAY. BEAS-2B CELLS WERE TREATED WITH RES, CSE AND TRANSFECTED WITH MIR-34A-5P MIMICS. CELLULAR SENESCENCE WAS EVALUATED BY SENESCENCE -RELATED BETA-GALACTOSIDASE (SA-BETA-GAL) STAINING AND EXPRESSION OF SENESCENCE-RELATED GENES (P16, P21, AND P53). THE EXPRESSIONS OF MIR-34A-5P, SIRT1, AND NF-KAPPAB P65 WERE EXAMINED USING QUANTITATIVE REAL TIME POLYMERASE CHAIN REACTION AND WESTERN BLOTTING. THE SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE (SASP) CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) WERE ASSESSED BY ENZYME-LINKED IMMUNOSORBENT ASSAY. THE BINDING BETWEEN MIR-34A-5P AND SIRT1 WAS CONFIRMED BY DUAL-LUCIFERASE REPORTER ASSAY. THE RESULTS SHOWED THAT CSE DOSE-DEPENDENTLY DECREASED CELL VIABILITY AND ELEVATED CELLULAR SENESCENCE, CHARACTERIZED BY INCREASED SA-BETA-GAL STAINING AND SENESCENCE-RELATED GENE EXPRESSIONS (P16, P21, AND P53). FURTHER, CSE DOSE-DEPENDENTLY INCREASED THE EXPRESSION OF MIR-34A-5P AND SASP CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) IN BEAS-2B CELLS. PRETREATMENT WITH RES INHIBITED CSE-INDUCED CELLULAR SENESCENCE AND SECRETION OF SASP CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) IN A DOSE-DEPENDENT MANNER. MOREOVER, RES REVERSED THE CSE-INDUCED DOWN-REGULATION OF SIRT1 AND UP-REGULATION OF MIR-34A-5P AND NF-KAPPAB P65. SIRT1 IS A TARGET OF MIR-34A-5P. OVEREXPRESSION OF MIR-34A-5P VIA TRANSFECTION WITH MIR-34A-5P MIMIC IN BEAS-2B CELLS ATTENUATED THE INHIBITORY EFFECT OF RES ON CELLULAR SENESCENCE, ACCOMPANIED BY REVERSING THE EXPRESSION OF SIRT1 AND NF-KAPPAB P65. IN CONCLUSION, RES ATTENUATED CSE-INDUCED CELLULAR SENESCENCE IN BEAS-2B CELLS BY REGULATING THE MIR-34A/SIRT1/NF-KAPPAB PATHWAY, WHICH MAY PROVIDE A NEW APPROACH FOR COPD TREATMENT. 2022 19 392 38 AN ORALLY BIOAVAILABLE CHEMICAL PROBE OF THE LYSINE METHYLTRANSFERASES EZH2 AND EZH1. EZH2 OR EZH1 IS THE CATALYTIC SUBUNIT OF THE POLYCOMB REPRESSIVE COMPLEX 2 THAT CATALYZES METHYLATION OF HISTONE H3 LYSINE 27 (H3K27). THE TRIMETHYLATION OF H3K27 (H3K27ME3) IS A TRANSCRIPTIONALLY REPRESSIVE POST-TRANSLATIONAL MODIFICATION. OVEREXPRESSION OF EZH2 AND HYPERTRIMETHYLATION OF H3K27 HAVE BEEN IMPLICATED IN A NUMBER OF CANCERS. SEVERAL SELECTIVE INHIBITORS OF EZH2 HAVE BEEN REPORTED RECENTLY. HEREIN WE DISCLOSE UNC1999, THE FIRST ORALLY BIOAVAILABLE INHIBITOR THAT HAS HIGH IN VITRO POTENCY FOR WILD-TYPE AND MUTANT EZH2 AS WELL AS EZH1, A CLOSELY RELATED H3K27 METHYLTRANSFERASE THAT SHARES 96% SEQUENCE IDENTITY WITH EZH2 IN THEIR RESPECTIVE CATALYTIC DOMAINS. UNC1999 WAS HIGHLY SELECTIVE FOR EZH2 AND EZH1 OVER A BROAD RANGE OF EPIGENETIC AND NON-EPIGENETIC TARGETS, COMPETITIVE WITH THE COFACTOR SAM AND NON-COMPETITIVE WITH THE PEPTIDE SUBSTRATE. THIS INHIBITOR POTENTLY REDUCED H3K27ME3 LEVELS IN CELLS AND SELECTIVELY KILLED DIFFUSED LARGE B CELL LYMPHOMA CELL LINES HARBORING THE EZH2(Y641N) MUTANT. IMPORTANTLY, UNC1999 WAS ORALLY BIOAVAILABLE IN MICE, MAKING THIS INHIBITOR A VALUABLE TOOL FOR INVESTIGATING THE ROLE OF EZH2 AND EZH1 IN CHRONIC ANIMAL STUDIES. WE ALSO DESIGNED AND SYNTHESIZED UNC2400, A CLOSE ANALOGUE OF UNC1999 WITH POTENCY >1,000-FOLD LOWER THAN THAT OF UNC1999 AS A NEGATIVE CONTROL FOR CELL-BASED STUDIES. FINALLY, WE CREATED A BIOTIN-TAGGED UNC1999 (UNC2399), WHICH ENRICHED EZH2 IN PULL-DOWN STUDIES, AND A UNC1999-DYE CONJUGATE (UNC2239) FOR CO-LOCALIZATION STUDIES WITH EZH2 IN LIVE CELLS. TAKEN TOGETHER, THESE COMPOUNDS REPRESENT A SET OF USEFUL TOOLS FOR THE BIOMEDICAL COMMUNITY TO INVESTIGATE THE ROLE OF EZH2 AND EZH1 IN HEALTH AND DISEASE. 2013 20 690 26 BRD4 DEGRADATION BLOCKS EXPRESSION OF MYC AND MULTIPLE FORMS OF STEM CELL RESISTANCE IN PH(+) CHRONIC MYELOID LEUKEMIA. IN MOST PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) CLONAL CELLS CAN BE KEPT UNDER CONTROL BY BCR::ABL1 TYROSINE KINASE INHIBITORS (TKI). HOWEVER, OVERT RESISTANCE OR INTOLERANCE AGAINST THESE TKI MAY OCCUR. WE IDENTIFIED THE EPIGENETIC READER BRD4 AND ITS DOWNSTREAM-EFFECTOR MYC AS GROWTH REGULATORS AND THERAPEUTIC TARGETS IN CML CELLS. BRD4 AND MYC WERE FOUND TO BE EXPRESSED IN PRIMARY CML CELLS, CD34(+) /CD38(-) LEUKEMIC STEM CELLS (LSC), AND IN THE CML CELL LINES KU812, K562, KCL22, AND KCL22(T315I) . THE BRD4-TARGETING DRUG JQ1 WAS FOUND TO SUPPRESS PROLIFERATION IN KU812 CELLS AND PRIMARY LEUKEMIC CELLS IN THE MAJORITY OF PATIENTS WITH CHRONIC PHASE CML. IN THE BLAST PHASE OF CML, JQ1 WAS LESS EFFECTIVE. HOWEVER, THE BRD4 DEGRADER DBET6 WAS FOUND TO BLOCK PROLIFERATION AND/OR SURVIVAL OF PRIMARY CML CELLS IN ALL PATIENTS TESTED, INCLUDING BLAST PHASE CML AND CML CELLS EXHIBITING THE T315I VARIANT OF BCR::ABL1. MOREOVER, DBET6 WAS FOUND TO BLOCK MYC EXPRESSION AND TO SYNERGIZE WITH BCR::ABL1 TKI IN INHIBITING THE PROLIFERATION IN THE JQ1-RESISTANT CELL LINE K562. FURTHERMORE, BRD4 DEGRADATION WAS FOUND TO OVERCOME OSTEOBLAST-INDUCED TKI RESISTANCE OF CML LSC IN A CO-CULTURE SYSTEM AND TO BLOCK INTERFERON-GAMMA-INDUCED UPREGULATION OF THE CHECKPOINT ANTIGEN PD-L1 IN LSC. FINALLY, DBET6 WAS FOUND TO SUPPRESS THE IN VITRO SURVIVAL OF CML LSC AND THEIR ENGRAFTMENT IN NSG MICE. TOGETHER, TARGETING OF BRD4 AND MYC THROUGH BET DEGRADATION SENSITIZES CML CELLS AGAINST BCR::ABL1 TKI AND IS A POTENT APPROACH TO OVERCOME MULTIPLE FORMS OF DRUG RESISTANCE IN CML LSC. 2022